Biosensors, method for obtaining the same and uses thereof

ABSTRACT

The invention concerns biosensors, the method for obtaining them and their uses in particular for detecting, assaying or locating, in direct immunofluorescence, a ligand such as an antigen or a hapten, in a heterogeneous population. Said biosensors consist of (i) at least a protein-type receptor fragment, capable of binding with a suitable ligand via an active site and in which fragment, at least one of its amino acid residues, located in the proximity of said active site is naturally present in the form of a Cys residue or is substituted in Cys residue, and (ii) a fluorophore coupled with said Cys residue(s).

[0001] The present invention relates to biosensors, to the method forobtaining the same and to uses thereof, in particular for detecting,assaying or locating, by direct immunofluorescence, a ligand such as anantigen or a hapten, in a heterogeneous population.

[0002] A biosensor is a bifunctional biological macromolecule: firstly,it is capable of specifically binding its ligand in a mixture; secondly,it translates the binding event into a signal which can be measureddirectly and instantaneously: by means of a simple system of diodes, ofa spectrofluorimeter, of a fluorescence microscope, of a confocalmicroscope, or of any other device, the consequence of which being thatthe detection thus carried out is immediate.

[0003] Although DNA “chips” for detecting nucleic acid sequences alreadyexist, a general solution for constructing biosensors and chips based onproteins, allowing the detection of other ligands, does not exist andtherefore remains to be developed.

[0004] Several approaches to this have been tried:

[0005] in the case of proteins which do not have cysteine (Cys) residuesor, a fortiori, disulfide bridges, a cysteine has been introduced intothe protein by site-directed mutagenesis. The cysteine is introducedeither in the immediate proximity of the ligand-binding site (proteinGB1; Sloan et al., 1998) or at a distance from this site (MBP protein;Gilardi et al., 1994; Marvin et al., 1997; GBP protein; Marvin et al.,1998; Tolosa et al., 1999). The thiol function thus added allowscoupling to a fluorophore sensitive to its electron environment. Thebinding of the ligand then modifies this environment and the change inthe properties of the biosensor is thus directly detectable byspectrofluorimetry. For example, Sloan et al. (1998) have in particularshown that the fluorophore should be positioned in a region located atthe protein-protein interface, and they have studied the coupling of afluorophore in a region of this type, for the GB1 domain-Fc complex: inthis context, the formation of the complex is analyzed; the best resultsare obtained when the fluorophore is coupled at an amino acid involvedin the formation of the complex.

[0006] However, this approach concerns only very particular proteinswhich have no free cysteine residue and no cysteine residues involved indisulfide bridges. Moreover, the use of the abovementioned biosensors islimited to the detection of a few particular ligands (maltose, glucoseand antibody Fc fragment). In fact, in this approach, the maindifficulty in creating biosensors, using a protein which containsdisulfide bridges, comes from the fact that the steps for coupling thefluorophore to a Cys residue may result in disulfide bridges, which areessential for the structure and stability, being attacked and themolecule being inactivated. Among these proteins, antibodies representthe class of proteins naturally dedicated to the specific binding ofprotein, peptide, polysaccharide or hapten ligands (antigens) with greataffinity. However, antibody molecules have Cys residues which formintra- and interchain disulfide bridges. In particular, the Fv fragmentsof antibodies comprise two disulfide bridges, one in each of the VH andVL domains, which are essential for the stability of the Fv fragment orof single-chain fragments, scFv (Glockshuber et al., 1992).

[0007] a biosensor has been derived from an antibody which binds2,4-dinitrophenol, using the following approach. A thiol group wasintroduced in the proximity of the ligand-binding site using an affinitylabel which combined, in the same molecule, a 2,4-dinitrophenol grouprecognized specifically by the antibody, a thiophenyl or disulfidelinkage, and an aldehyde or alpha-bromoketone reactive group. Theaffinity label was capable of binding to the binding site of theantibody via the 2,4-dinitrophenol group, and then of coupling to theantibody by random reaction of its aldehyde or alpha-bromoketone groupwith the side chains of the Lys, His or Tyr residues located nearby.Treatment of the coupled antibody with dithiothreitol liberated an SHgroup which could, in turn, be coupled to a fluorophore (Pollack et al.,1988).

[0008] This method has the drawback that it is random, cannot begeneralized, and applies only to antibodies directed against haptenssince it depends on the existence of Lys, His or Tyr residues at acorrect distance from the binding site of the antibody and on thepossibility of synthesizing a three-part affinity label as describedabove.

[0009] The inventors have given themselves the aim of enablingprotein-based biosensors to be obtained using a rational, general andgeneralizable method capable of identifying the amino acid residues of aprotein receptor which may be substituted with cysteine residues. Suchbiosensors correspond better to practical needs, in particular in thatthey allow coupling of a fluorophore at a position which induces achange in the structural environment of the fluorophore during ligandbinding, regardless of whether the crystalline structure of the complexbetween the receptor and the ligand is known.

[0010] Such biosensors can be constructed using any type of protein, inparticular proteins having cysteine residues which are important fortheir structure or their stability, and constitute novel tools fordetecting, assaying and locating a large variety of ligands in aheterogeneous population of molecules.

[0011] A subject of the present invention is a biosensor, characterizedin that it consists of (i) at least one fragment of a receptor which isprotein in nature, capable of binding to a suitable ligand via an activesite, and in which fragment at least one of its amino acid residueslocated in the proximity of said active site is naturally present in theform of a Cys residue, or is substituted with a Cys residue, and (ii) afluorophore coupled to said Cys residue(s).

[0012] The Cys residues which are not essential, for example those whichare located far from the active site, can be changed into other residuesby site-directed mutagenesis, in particular into Ser or Ala residues, soas to avoid interference coupling with the fluorophores.

[0013] For the purpose of the present invention, the term “receptor” isintended to mean a protein macromolecule having an active site, capableof binding a ligand, in particular an antibody, a hormone or bacterialreceptor, an affinity protein, a transport protein or a viral receptoror else any polypeptide having a specific affinity for a ligand.

[0014] For the purpose of the present invention, the term “ligand” isintended to mean any molecule capable of binding to said receptor viasaid active site, in particular a protein, peptide or hapten antigen,such as a bacterial antigen, or a hormone, a cytokine, an interleukin,TNF (Tumor Necrosis Factor), a growth factor, a viral protein, or apeptide or nucleotide sequence.

[0015] For the purpose of the present invention, the expression “activesite of the receptor or of the receptor fragment” is intended to meanall of the residues which contribute to the binding of the ligand. Thisactive site is also called binding site or paratope.

[0016] For the purpose of the present invention, the term “proximity” isunderstood to be as defined by the mathematical theory of topologicalspaces; the residues of the receptor which are in the proximity of theactive site are the residues which are in direct contact with theligand, those which are in contact by water molecules, and those forwhich the solvent accessible surface area (ASA; Creighton, 1993) ismodified by the binding of the ligand. The use of spheres of increasingradius, from 1.4 to a maximum of 30 Å, preferably from 1.4 to 2.9 Å,that is to say greater than that of a water molecule (1.4 Å), forexample 1.4, 1.7, 2.0, 2.3, 2.6 and 2.9 Å, to calculate the solventaccessible surface area, makes it possible to define an increasinglylarge proximity for the binding site of the receptor, and thus toincrease the set of potential sites for coupling the fluorophore, takinginto account the considerable volume of a fluorophore compared to thatof a water molecule. The mutation to cysteine must not be greatlydeleterious to the interaction with the ligand if the aim is for thelabeled receptor to maintain good affinity for the ligand.

[0017] For the purpose of the present invention, the term “fluorophore”is intended to mean any molecule the fluorescence of which is sensitiveto its microenvironment, and which can be coupled to a Cys residue.

[0018] According to an advantageous embodiment of said biosensor, saidreceptor has one or more disulfide bridges essential to its activity orto the maintaining of its structure.

[0019] According to an advantageous embodiment of said biosensor, saidreceptor is an antibody or an antibody fragment, such as an Fv, scFv orFab fragment or a miniantibody; said antibody is advantageously anatural or artificial monoclonal antibody.

[0020] In accordance with the invention, the fluorophore is inparticular selected from the group consisting of: IANBD, CNBD,acrylodan, 5-iodoacetamidofluorescein (5-IAF) or a fluorophore having analiphatic chain of 1 to 6 carbon atoms.

[0021] According to another advantageous embodiment of said biosensor,the biosensor is in soluble form or is immobilized on a suitable solidsupport made of plastic material or glass.

[0022] According to yet another advantageous embodiment of saidbiosensor, said solid support is a microplate or an optical fiber.

[0023] Among the immobilization methods, mention may be made of thosedescribed in Piervincenzi et al., 1998; Yoshioka et al., 1991; Turkova,1999; Saleemuddin, 1999; Weetall, 1993; Sara et al., 1996.

[0024] When the support is an optical fiber, such biosensors may beimplanted in situ, for example into a vein of a patient or of an animal,or into an individual cell, so as to assay the ligand in vivo,continuously, and thus to follow its kinetics of appearance anddisappearance.

[0025] The subject of the present invention is also a protein-basedchip, characterized in that it consists of a solid support on which atleast one biosensor according to the invention is immobilized.

[0026] Such protein-based chips comprise a solid support, for example amicroplate, on which the molecules of various biosensors areadvantageously immobilized in a matrix and included in microfluidcircuits. Such biosensors make it possible to simultaneously andinstantaneously detect and assay a large number of ligands in a sample,for example a sample of body fluid.

[0027] The biosensors according to the invention have a certain numberof advantages:

[0028] the residue of the receptor on which the coupling of thefluorophore is carried out is predetermined and is a Cys residue locatedin the proximity of the active site of the receptor.

[0029] the coupling of the fluorophore on predetermined cysteineresidues is carried out under conditions in which it does not attack thedisulfide bridges possibly present on the receptor.

[0030] A subject of the present invention is also a method for preparingbiosensors as defined above, characterized in that it comprises thefollowing steps:

[0031] (a) selecting residues of the receptor by searching for theresidues which, in the receptor-ligand complex, (i) are in directcontact with the ligand, or (ii) are in contact via a water molecule, or(iii) have a solvent accessible surface area (ASA) which is modified bythe binding of the ligand, when use is made of spheres of increasingradius of 1.4 to 30 Å, preferably of 1.4 to 2.9 Å, for the molecule ofsaid solvent;

[0032] (b) calculating the solvent accessible surface area (ASA), forthe free receptor, of the atoms in the γ position and, optionally, inthe δ position for each amino acid residue selected in (a), using asphere of radius 1.4 Å (corresponding to a water molecule), andselecting the residues in which the atom in the γ position or the atomin the δ position is accessible to the solvent; the solventaccessibility of an atom in the δ position is informative with respectto that of the Sγ of a corresponding Cys residue. Conventionally, theaccessibility threshold is fixed at a minimum of 2%, preferably at20-25%, and more favorably greater than 25%;

[0033] (c) mutating by site-directed mutagenesis at least one of theresidues selected in (b) to a Cys residue when said residue is notnaturally a Cys residue, and

[0034] (d) coupling the Sγ atom of at least one Cys residue obtained in(b) or (c) to a fluorophore. In this case, the Sγ atom of the Cysresidue, which may or may not be a mutant residue, which is exposed tothe solvent is the target for the fluorophore molecule. It is consideredthat the Sγ of the Cys residue, which may or may not be a mutantresidue, superposes with the atom in the γ position of the wild-typeresidue. This atom in the γ position must therefore be exposed to thesolvent in the structure of the wild-type receptor. When an atom ispresent in the δ position in the wild-type residue, it may mask the atomin the γ position, with respect to the solvent, whereas this maskingwill no longer exist after it has been changed into Cys.

[0035] According to an advantageous embodiment of said method, in step(b), the ASA values for a residue X_(i) in the i position of thesequence of the receptor are expressed in the form of percentages of thecorresponding ASA values in a tripeptide Gly-X_(i)-Gly, which wouldadopt the same configuration as the tripeptide X_(i−1)-X_(i)-X_(i+1) inthe structure of the receptor.

[0036] The residues selected in step (b) are divided into three classes:

[0037] the first class contains the residues in which the atom in the γposition is accessible to the solvent in the structure of the freereceptor, and which are in contact with the ligand either directly orvia a water molecule;

[0038] the second class contains the residues in which the atom in the γposition is accessible and which are not in direct contact with theligand;

[0039] the third class contains the residues in which the atom in the δposition is accessible but not the atom in the γ position.

[0040] When the crystalline structure of one of the partners of thereceptor/ligand pair, or of the complex thereof, is unknown orunavailable, in this case, said method comprises, prior to step (a) asdefined above, a step of modeling the molecule for which the structureis unknown or unavailable: receptor or active fragment of this receptor(for example an antibody Fv fragment), its ligand (antigen), or thecomplex (receptor-ligand) thereof.

[0041] Advantageously, said modeling step is carried out via acomputer-assisted method; mention may be made, for example, of themethods described in Sternberg, 1996; Rees et al., 1996.

[0042] As a variant, a subject of the present application is a methodfor preparing biosensors, which comprises the following steps:

[0043] (a₁) identifying the active site of the receptor by mutagenesisof the set, or of a subset, of the residues of the receptor, anddetermining the variations in the parameters of interaction with theligand (K_(D), k_(on), k_(off)) which are due to each mutation or tolimited groups of mutations;

[0044] (b₁) selecting the Cys residues, or the residues to be mutated tocysteine, from the residues of the receptor which are located in theproximity of the residues of the active site along the sequence;

[0045] (c₁) mutating by site-directed mutagenesis at least one of theresidues selected in (b₁) to a Cys residue when said residue is notnaturally a Cys residue; and

[0046] (d₁) coupling the Sγ atom of at least one Cys residue obtained in(b₁) or in (c₁) to a fluorophore.

[0047] For example, the sequences of the hypervariable loops of theantibody mAbD1.3, named CDRs (Complementary Determining Regions orregions which determine antibody-antigen complementarity), can bedefined using sequence comparisons (England et al., 1999). The activesite of mAbD1.3 for its interaction with lysozyme has been characterizedby changing the residues of the CDRs, mainly to Ala (Dall'Acqua et al.,1996, 1998; England et al., 1997, 1999; Ito et al., 1993, 1995; Hawkinset al., 1993; Ysern et al., 1994; Goldbaum et al., 1996). The light (L)chain residue changes L-H30A, L-Y32A, L-Y49A, L-Y50A and L-Y92A increasethe dissociation constant for the Fv or scFv fragment of mAbD1.3 andlysozyme, whereas the changes L-I29T, L-T51A, L-T52K, L-T53A, L-S93A andL-T97S affect it very little or not at all. Similarly, the heavy (H)chain residue changes H-F27N, H-Y32A, H-W52A, H-D54A, H-D100A, H-Y101A,H-R102K or H-R102M increase the dissociation constant whereas H-T30A,H-N56A, H-D58A and H-R99A affect it very little or not at all.

[0048] Consequently, the residues L-Thr51, L-Thr52, L-Thr53, L-Ser93,L-Thr94, H-Thr30, H-Asn56, H-Asp58 and H-Arg99, which are polar andtherefore probably exposed at the surface of the antibody mAbD1.3 andwhich are close to the residues of the active site in the sequence,would constitute potential coupling sites according to purely functionalcriteria. Certain common potential sites are indeed found when criteriabased on structure (points (a) and (b) of the general method, above) oron function (points (a₁) and (b₁) of the variant of said method, above)are used, for example L-Thr52, L-Thr53, L-Ser93, H-Thr30, H-Asn56,H-Asp58 and H-Arg99.

[0049] According to another advantageous embodiment of said method,prior to step (a) or to step (a₁), the nonessential Cys residues of thereceptor are substituted with Ser or Ala residues by site-directedmutagenesis.

[0050] According to another advantageous embodiment of said method, instep (d) or in step (d₁), said fluorophore is selected from the groupconsisting of IANBD, CNBD, acrylodan, 5-iodoacetamidofluorescein or afluorophore having an aliphatic chain of 1 to 6 carbon atoms.

[0051] According to another advantageous embodiment of said method,prior to step (d) or to step (d₁), the mutated receptor obtained in step(c) or in step (c₁) is subjected to a controlled reduction; in fact, thecoupling yield may be significantly improved when the step is added.

[0052] According to yet another advantageous embodiment of said method,after step (d) or step (d₁), it comprises an additional step (e) or (e₁)for purifying the biosensor, for example on an exclusion or affinitycolumn, in particular an immobilized nickel ion column when the receptorfragment or antibody fragment comprises a His-residue extension.

[0053] According to yet another advantageous embodiment of said method,after step (e) or step (e₁), it comprises an additional step formeasuring the equilibrium constant (K_(D) or K′_(D)) or the dissociation(K_(off)) and association (k_(on)) rate constants for the receptor andligand.

[0054] The equilibrium constant is named K_(D) when the complex is insolution and K′_(D) when one of the partners of the complex isimmobilized.

[0055] Said constant may, for example, be determined from measurementsof fluorescence by intensity or by anisotropy. All the data obtained(K_(D), K′_(D), k_(off), k_(on) size of change in fluorescence duringligand binding, shift of emission maximum) make it possible to refinethe selection of the residues of the receptor which are the mostsuitable for coupling the fluorophore.

[0056] According to yet another advantageous embodiment of said method,after step (d) or (d₁) or step (e) or (e₁), it comprises an additionalstep for immobilizing the biosensor on a suitable solid support asmentioned above.

[0057] A subject of the present invention is also the use of saidbiosensors, alone or as a mixture, for applications comprisingdetecting, assaying and locating ligands.

[0058] The biosensors according to the invention make it possible, forexample, to detect, assay or locate an antigen or a hapten in aheterogeneous population of molecules, instantaneously.

[0059] In the health domain, said biosensors are of use:

[0060] for monitoring the progression or the regression of a disease, inresponse to a treatment;

[0061] for assaying an infectious agent (bacterium, virus), a pathogenicagent (tumor cell), a macromolecule (hormone, cytokine) or a hapten in abody fluid (for example blood, sperm, etc.);

[0062] for determining the serotype of an infectious agent;

[0063] for detecting a cellular marker and determining its location;

[0064] for sorting cells, on the basis of the presence of a surfacemarker;

[0065] for sorting molecules;

[0066] for quantifying an intracellular or extracellular component,monitoring its kinetics of appearance and disappearance and monitoringits location;

[0067] for determining the half-life of a molecule (depending on itsinstability, on its metabolism or on its elimination), its diffusion andits tissue concentration;

[0068] for monitoring the effect of a chemical molecule on a cellularcomponent (screening libraries, in particular protein libraries).

[0069] In the domain of the environment, of industry and of suppressionof fraud, said biosensors are also of use:

[0070] for detecting and assaying a pollutant, and monitoring itselimination, by natural bioremediation processes;

[0071] for assaying an active component in a preparation being producedor marketed;

[0072] for monitoring the kinetics of a chemical synthesis reaction.

[0073] Said biosensors are also of use in producing protein-based chips.

[0074] The subject of the present invention is also reagents fordetecting, assaying and/or locating ligands, characterized in that theycomprise at least one biosensor as defined above.

[0075] A subject of the present invention is also a method fordetecting, assaying or locating a ligand in a heterogeneous sample,characterized in that it comprises bringing said heterogeneous sampleinto contact with at least one reagent according to the invention.

[0076] A subject of the present invention is also a kit for detecting,assaying and/or locating ligands, characterized in that it includes atleast one reagent according to the invention.

[0077] A subject of the invention is also a kit for screening forinhibitors of the ligand/receptor interaction, characterized in that itincludes at least one biosensor according to the invention.

[0078] A subject of the present invention is also the use of the plasmidpMR1 of sequence SEQ ID NO: 10, deposited with the Collection Nationalede Culture de Mircoorganismes (CNCM) [National Collection of Culturesand Microorganisms], under the number I-2386, dated Feb. 29, 2000, forpreparing a biosensor in accordance with the invention.

[0079] A subject of the present invention is also the use of the plasmidpMR1(VL-S93C) of sequence SEQ ID NO: 11, deposited with the CollectionNationale de Culture de Microorganismes (CNCM), under the number I-2387,dated Feb. 29, 2000, for preparing a biosensor in accordance with theinvention.

[0080] A subject of the present invention is also the plasmid pMR1 ofsequence SEQ ID NO: 10, deposited with the Collection Nationale deCulture de Microorganismes (CNCM), under the number I-2386, dated Feb.29, 2000.

[0081] A subject of the present invention is also the plasmid pMR1(VL-S93C) of sequence SEQ ID NO: 11, deposited with the CollectionNationale de Culture de Microorganismes (CNCM) under the number I-2387,dated Feb. 29, 2000.

[0082] Besides the above arrangements, the invention also comprisesother arrangements, which will emerge from the following description,which refers to examples of implementation of the method which is thesubject of the present invention, and also to the attached drawings, inwhich:

[0083]FIG. 1 illustrates the criteria for determining the residues ofthe Fv fragment of the antibody named mAbD1.3, in the proximity of theactive site for binding to lysozyme, which can be used for coupling thefluorophore. Columns 1 and 2 indicate the residues of the Fv fragment ofthe antibody named mAbD1.3 which are in direct contact with thelysozyme, either at least via their side chain (d), or via their peptidechain (b); those which are in contact via water molecules (i), and thosein which the solvent accessible surface area (ASA) is modified by thebinding of the lysozyme when spheres of radius 1.4, 1.7, 2.0, 2.3, 2.6or 2.9 Å are used for the molecule of solvent. Columns 3 and 4 indicatethe ASA values, in the free Fv (using R=1.4 Å), of the atoms in the γposition and, optionally, δ position of each of the residues X_(i)selected, in the i position of the sequence of the Fv, in the form ofpercentages of the corresponding ASAs in a tripeptide Gly-X_(i)-Glywhich would adopt the same conformation as the tripeptideX_(i−1)-X_(i)-X_(i+1) in the structure of the Fv. Columns 5, 6 and 7indicate the ASA, in the structure of the native complex, of the groupsin the γ, δ and ε positions in the initial side chain. Columns 8 and 9indicate the mutations of these residues known in mAbD1.3 and theireffects, in the form of a variation ΔΔG of energy of interaction withlysozyme. Column 10 summarizes the classification of the residues inorder of decreasing priority; (+) corresponds to the residues in whichthe atom in the γ position is accessible to the solvent in the structureof the free Fv, and which are in contact with the lysozyme eitherdirectly or via a water molecule in the structure of the complex, (+/−)corresponds to the residues in which the atom in the γ position isaccessible and which are not in direct contact with the lysozyme, and(?) corresponds to the residues in which the atom in the δ position isaccessible but not the atom in the γ position.

[0084]FIG. 2 illustrates the optimization of the production ofrecombinant mAbD1.3 Fv (scFv-His6) using prokaryotic expression vectors(pMR1 and pMR5). The periplasmic content of E. Coli strains HB2151 andBL21 (DE3) transformed, respectively, with the plasmids pMR5 and pMR1,induced by IPTG (BL21 (DE3)) or anhydrotetacyclin (HB2151) and culturedwhile varying the temperature, the induction time and the inducerconcentration, was measured by ELISA using an anti-His5 antibody. Theresults are expressed in arbitrary units.

[0085]FIG. 3 illustrates the analysis, by SDS-polyacrylamide gelelectrophoresis and Coomassie blue staining, of the fractions from thepurification of the mutant scFv-His6(VL-S93C) on a nickel ion column.EP: unpurified periplasmic extract, NR: fractions not retained, M:molecular mass marker, L₂₀ and L₄₀ fractions from washing with 20 and 40mM of imidazole, EL: elution fractions.

[0086]FIGS. 4 and 5 illustrate the noncovalent oligomerization of thewild-type scFv-His6 and of the VL-S93C mutant, analyzed by sizeexclusion chromatography. The purified scFv-His6s were injected at aconcentration of 50 μg/ml for the wild-type and for the mutant, in avolume of 200 μL, at the top of a Superdex^(R) 75 HR 10/30 column(Pharmacia). The chromatogram was developed at 25° C., at pH=7.5 for thewild-type and at pH=7.0 for the mutant.

[0087]FIG. 6 represents the chemical structures of CNBD, of IANBD, of5-iodoacetamidofluorescein (5-IAF) and of acrylodan.

[0088]FIG. 7 illustrates the proportionality between fluorescenceintensity (excitation at 469 nm, emission at 535 nm) and proteinconcentration in the elution fractions derived from the separationbetween conjugate and free fluorophore.

[0089]FIG. 8 illustrates the fluorescence emission by scFv-Hisδderivatives, for an excitation at 469 nm, in the absence of lysozyme.The following species were analyzed: wild-type scFv-His6 andscFv-His6(VL-S93C) after coupling with IANBD and separations; sFv-His6(VL-S93C) before coupling.

[0090]FIG. 9 shows the evolution of the coupling yield (proportion ofmutant scFv-His6s linked to a fluorophore, here IANBD) and of thematerial yield when the biosensor is prepared, as a function of theconcentration of 2-mercaptoethanol used for the controlled reduction.

[0091]FIG. 10 shows the absorption spectrum of the fluorescentderivative scFv-His6(V-S93ANBD), recorded for wavelengths of 250 to 550nm. The height of the peak at 278 nm, corresponding to the protein, andthat of the peak at 500 nm, corresponding to the coupled IANBD, made itpossible to calculate the coupling yield (here 83%).

[0092]FIG. 11 illustrates the fluorescence emission by thescFv-His6(VL-S93ANBD) derivative for an excitation at 469 nm expressedin arbitrary units (a.u.). The spectra were recorded in the absence oflysozyme, or 1 minute after addition of lysozyme at 100 nM and 1 μM(final concentrations) and brief homogenization. The same maximumenhancement of fluorescence (+27%) is observed for three differentpreparations of this same derivative, for which various coupling methods(with or without prior reducing treatment) were used, and variouscoupling yields were obtained.

[0093]FIG. 12 illustrates the fluorescence emission at 535 nm by thescFv-His6(VL-S93ANBD) conjugate, for an excitation at 480 nm, expressedin arbitrary units (a.u.). The fluorescence intensities were recorded inthe absence or in the presence of varying concentrations of lysosyme (10nM to 2 μM) in a 50 mM Tris-HCl, 150 mM NaCl buffer, pH=7.5. In thisbuffer, the fluorescence intensity of the scFv-His6(VL-S93ANBD)conjugate is proportional to the concentration of lysozyme up to 400 nMand increased by 91% at saturation. The curve obtained makes it possibleto directly titrate the lysozyme between 10 and 400 nM.

[0094]FIG. 13 illustrates the coupling yields and the fluorescenceproperties of the biosensors. Column 1 indicates the residues of the Fvfragment of the antibody named mAbD1.3 which have been mutated to Cysresidues; these residues were chosen as a function of the structuraldata set out in FIG. 1. Column 2 indicates the percentage of moleculesof the mutant scFv-His6 which are coupled to the IANB, compared with thewild-type (wt) scFv-His6. Column 3 indicates the variation influorescence at 535 nm between the free forms and the forms complexedwith lysozyme of the scFv-Cys-ANBD conjugates, measured in a 20 mMTris-HCl, 500 mM NaCl, 160 mM imidazole buffer, pH=7.9. Considerableenhancements of fluorescence are obtained for more than half thecoupling positions.

[0095] It should be clearly understood, however, that these examples aregiven only by way of illustration of the subject of the invention, ofwhich they in no way constitute a limitation.

EXAMPLE 1 Materials and Methods

[0096] 1—Parental Bacterial Strains, Plasmids and Phages TABLE 1 StrainMain characteristics Reference HD2151 Ara Δ(lac-pro) thi/F′ Carter etal., 1985 proA⁺B⁺ lacI^(q)ZΔM15 RZ1032 Hfr dut ung r⁺ m⁺ Kunkel et al.,1987 BL21 (DE3) F⁻ ompT hsdS_(B)(r_(B) ⁻, m_(B) ⁻) Studier and Moffatt,gal dcm/lambdaDE3 1986

[0097] The vector pASK98-D1.3 encodes a hybrid between an scFv of themonoclonal antibody mAbD1.3 and a tag of the streptavidin type. Thecorresponding gene is under the control of the tetracycline promoter andoperator (Skerra, 1994). pSPI1.0 is a derivative of pET26b (Novagen) andallows scFvs to be subcloned under the control of the T7 phage promoter.

[0098] 2—Culture Media and Buffers

[0099] LB and 2xYT media have been described (Sambrook et al., 1989). SBmedium contains 16 g/l bactotryptone, 10 g/l bactoyeast, 10 g/l NaCl, 50mM K₂HPO₄, 5 mM MgSO₄ and 1% glucose (Plückthun et al., 1996).Ampicillin is used at 200 μg/ml and kanamycin at 30 μg/ml in the growthmedia. PBS and TBE buffers have been described (Sambrook et al.,mentioned above). Buffer P: 20 mM Tris-HCl, pH 7.9; 500 mM NaCl. BufferQ: 50 mM Tris-HC₁, pH 7.5; 150 mM NaCl. Buffer C: 50 mM sodium phosphatepH 7.0; 150 mM NaCl.

[0100] 3—Oligonucleotides

[0101] The sequences of the oligonucleotides used for the mutagenesis orthe genetic constructs are given in the following table. The modifiedcodons are marked in bold for the mutagenic oligonucleotides. TABLE IIName Sequence pMR1-VL-H30C 5′-atggtacgatttattaaacattataagggtg-3′ SEQ IDNO:20 pMR1-VL-N31C 5′-catgctaaataacagtggatattcccacttg-3′SEQ ID NO:17pMR1-VL-Y49C 5′-agacgattccaacaacatatacactggtcctcga-3′ SEQ ID NO:21pMR1-VL-Y50C 5′-gattccaacaacatatctggtcctcgactcctcta-3′ SEQ ID NO:22pMR1-VL-T52C 5′-catctgctaaggtacatgtataatagac-3′ SEQ ID NO:13pMR1-VL-T53C 5′-catcgctaagcatgttgtataataga-3′ SEQ ID NO:1 pMR1-VL-D56C5′-cttgatggcacaccgcatgctaaggttgttg-3′ SEQ ID NO:16 pMR1-VL-W52C5′-gaggagtacgcaaaaatgttgacagtaa-3′ SEQ ID NO:12 pMR1-VL-S93C5′-cgaggagtacaccaaaaatgttgacagta-3′ SEQ ID NO:2 pMR1-VL-T94C5′-cgtccgaggacaactccaaaaatgtt-3′ SEQ ID NO:3 pMR1-VH-S28C5′-acaccatagccagttaagcagaaccctgagacg-3′ SEQ ID NO:18 pMR1-VH-G31C5′-caagagtaattggacaataccacatttg-3′ SEQ ID NO:24 pMR1-VH-T30C5′-acaccatagccgcataatgagaaccctg-3′ SEQ ID NO:15 pMR1-VH-Y32C5′-cagtttacaccacacccggttaatgaga-3′ SEQ ID NO:14 pMR1-VH-G53C5′-acccttactaaacacaactacctttgtgtctg-3′ SEQ ID NO:25 pMR1-VH-N56C5′-ttatagtctgtgcacccatcaccccaaatcat-3′ SEQ ID NO:19 pMR1-VH-R99C5′-tgacacggtctctcacactaatatccgaactgatga-3′ SEQ ID NO:23 Sequencing:VL-CDR1-Rev 5′-agaatattgtgttcctga-3′ SEQ ID NO:4 VH-CDR1-Rev5′-tgctgatgctcagtctgg-3′ SEQ ID NO:5 VH-Seq-CDR-35′-ggtgatggaaacacagac-3′ SEQ ID NO:6 pASK98-seq-His5′-cgccgcgcttaatgc-3′ SEQ ID NO:7 Construction of pMR1: pASK98-His6-For:5′-tcgagatcaagcggccgctggaacaccatcaccatcaccatta-3′ SEQ ID NO:8PASK98-His6-Back: 5′-agcttaatggtgatggtgatggtggtccagcggccgcttgatc-3′ SEQID NO:9

[0102] 4—Recombinant DNA Techniques

[0103] Preparation of plasmid DNA: the plasmid DNA minipreparations arecarried out using 5 ml of bacterial culture, by the alkaline lysismethod (Sambrook et al., mentioned above). The midipreparations arecarried out using 30 to 60 ml of culture, with the QIAFilter PlasmidMidikit (QIAgen).

[0104] Transformation: the preparation of competent bacteria by thesimple CaCl₂ method and the transformation by heat shock are carried outas described in Sambrook et al., mentioned above.

[0105] Purification of DNA fragments by electrophoresis: the restrictionmixture is loaded onto a 1 to 2% agarose gel (Easy Bag Agarose, QuantumBioprobe). The electrophoresis is performed for 1 to 2 hours at 8 V/cmin TBE buffer. The gel is then stained with ethidium bromide at 1 μg/mlin water. The agarose band containing the DNA fragment is cut out undera UV lamp and the DNA is then extracted and purified using the QIAquickGel Extraction Kit (QIAgen).

[0106] Ligation of a DNA fragment into a plasmid vector: for theligation of restriction fragments, the mixture thereof is precipitatedwith Precipitator (Appligene), resuspended in water and then brought to50° C. for 5 minutes. 400IU of T4 DNA ligase (New England Biolabs) andits buffer are added and the ligation is continued overnight at 16° C.The HB2151 strains are then transformed with the ligation products.

[0107] Mutagenesis: the site-directed mutagenesis is carried out asdescribed in Kunkel et al., (1987) using T4 polynucleotide kinase, T4DNA ligase and T4 DNA polymerase (New England Biolabs).

[0108] Sequencing: the presence of a mutation or the genetic constructsare verified by sequencing, using the T7-Sequencing Kit (Pharmacia),with the nucleotide mixtures for short reading, the dATP-³⁵S and aprimer. The matrix consists of a minipreparation of plasmid DNA,denatured with sodium hydroxide and then neutralized by passing it overa Microspin S-400-HR column. The sequence gels (6% acrylamide 1:19 bis,42% urea in TBE, 30×40 cm) are subjected to a preelectrophoresis for 45min at 40 W, and then loaded with the preboiled samples (Sambrook etal., mentioned above). The migration is performed at 40 W for 2 to 3 h.The gels are dried and exposed overnight with a BioMax MR film (Kodak).

[0109] 5—Recombinant Plasmid Constructions

[0110] pMR1: the vector pASK98-D1.3 is cleaved with the XhoI and HindIIIenzymes. The restriction mixture is passed over a Microspin S-400-HRcolumn so as to remove the smallest restriction fragment (43 bp). Theoligonucleotides pASK98-his6-for and pASK98-his6-back are phosphorylatedand hybridized at 60° C. so as to form a linker with sticky ends, whichis inserted into the large XhoI-HindIII fragment of pASK98-D1.3. Thephagemid obtained, pMR1, is controlled by restriction analysis with theNotI and PvuI enzymes and by sequencing using the oligonucleotidepASK98-seq-His6; it corresponds to SEQ ID NO: 10.

[0111] PMR 5: the plasmids pFBX and pSPI1.0 are cleaved with SfiI andNotI. The small fragment of pFBX (750 bp) containing the gene of theD1.3 scFv and the large fragment of pSPI1.0 (4650 bp) are purified onagarose gel and then assembled by ligation. The plasmid obtained, pMR5,is controlled by restriction analysis with the AflIII enzyme.

[0112] 6—Production of Antibody Fragments

[0113] Production of a small amount from pMR1 or pMR5: 200 ml of 2xYTampicillin medium are inoculated with an isolated colony of therecombinant strains HB2151(pMR1) or BL21(DE3, pMR5). The culture isshaken at 22, 30 or 37° C. until an A_(600 nm)=0.5 is obtained, at whichpoint it is induced with 0.2 to 1.0 μg/ml of anhydrotetracycline forHB2151 or with 0.2 to 1.0 mM of IPTG for BL21(DE3), and the shaking isthen continued for 2 h, 5 h or 16 h. A periplasmic extract is preparedby osmotic shock (see below) using 50 ml of culture centrifuged for 20min at 10,500 g, at 4° C.

[0114] Production of large amounts: to produce the scFv-His6s, SB medium(100 ml) supplemented with ampicillin is inoculated with an isolatedcolony of the recombinant strain HB2151 (pMR1). The culture is shakenovernight at 37° C. and then 25 ml of the contents are transferred into750 ml of the same medium preequilibrated at 22° C. The culture isshaken at 22° C. until an A_(600 nm)=2.0 is obtained, and is theninduced with 0.22 μg/ml of anhydrotetracycline. The growth is continuedfor approximately 2 h 30 min, until an A_(600 nm)=4 is obtained. Theculture is centrifuged for 20 minutes at 9500 g, at 4° C. Theperiplasmic extract is prepared using the polymyxin method describedbelow (example 1-7).

[0115] 7—Preparation of Periplasmic Extracts

[0116] The bacterial cultures are centrifuged and periplasmic extractsare prepared from the bacterial pellets using one of the following fourmethods. The volumes are indicated relative to the volume of culturetreated.

[0117] Osmotic shock method: the bacterial pellet is resuspended in 20%sucrose in 100 mM Tris HCl, pH 7.5 (1/20^(th) volume). The suspension ismaintained on ice for 10 minutes and then centrifuged for 15 minutes at10,000 g, at 4° C. The pellet is taken up in 0.5 mM MgCl₂ (1/20^(th)volume). The suspension is again maintained on ice for 10 minutes andcentrifuged in the same way. This second supernatant constitutes theactual periplasmic extract. The first supernatant may, however, be usedafter dialysis against buffer P.

[0118] Tris-EDTA method: the bacterial pellet is resuspended in 100 mMTris-HCl, pH 7.5; 1 mM EDTA (1/20^(th) volume). The suspension ishomogenized for 30 minutes by magnetic stirring at 4° C., and thencentrifuged for 30 minutes at 35,000 g. The supernatant constitutes theperiplasmic extract. The pellet is resuspended in 1/20^(th) volume ofSDS at 1% in water. It contains the insoluble, membrane-bound orcytoplasmic proteins.

[0119] Tris-EDTA-NaCl method: this method is identical to the Tris-EDTAmethod, except that the bacterial pellet is resuspended in 100 mMTris-HCl, pH 7.5; 1mM EDTA and 1 M NaCl.

[0120] Polymyxin method: this method is identical to the Tris-EDTA-NaClmethod, except for the single difference that the bacterial pellet isresuspended in buffer P containing 1 mg/ml of polymyxin B sulfate (ICN).

[0121] 8—Purification of Antibody Fragments

[0122] Purification of the scFv-His6s: the periplasmic extract isfiltered through a Millex filter (porosity of 0.45 μm, Millipore) andthen loaded onto a column containing 1.5 ml of Ni-NTA Superflow resin(QIAgen) equilibrated with 10 ml of buffer P. The column is successivelywashed with 10 ml of 5 mM imidazole, 20 mM imidazole and 40 mM imidazolein buffer P. The scFv-His6s are eluted with 4×1.5 ml of 100 mM imidazolein buffer P. The elution fractions are analyzed by SDS-15% PAGE,according to the protocol described in example 1-10. Their proteinconcentration is measured either by means of a Bradford reagent (BioRad)using bovine serum albumin (BSA) as the standard, or by means ofA_(280 nm), taking ε=51,130 M⁻¹.cm⁻¹, calculated as described in Pace etal. (1995).

[0123] 9—Quantification of scFv-His6 by Indirect ELISA

[0124] A microtitration plate (Nunc) is coated with hen egg whitelysozyme (HEL, 10 μg/ml) in 50 mM NaHCO₃ buffer, pH 9.6, and incubatedovernight at ambient temperature. The plate is saturated for 2 h at 37°C. with BSA (3% weight/volume) in PBS. The samples are diluted at least2-fold in PBS containing 3% BSA, loaded onto the plate and left atambient temperature for 1 h. The plate is washed 5 times with PBScontaining 0.05% Tween 20, and then incubated for 1 h in the presence ofa primary antibody (mouse anti-His5 monoclonal antibody, QIAgen) dilutedto 0.2 μg/ml in PBS containing 3% BSA. The plate is washed aspreviously, and then incubated for 1 h in the presence of a secondaryantibody (anti-mouse Fc monoclonal antibody coupled to alkalinephosphatase) diluted to 1/10,000. The plate is washed as previously andthe immunocomplexes are revealed with 2 mg/ml of p-nitrophenylphosphate(Sigma) in 1M diethanolamine-HCl, pH 9.8, 10 mM MgSO₄. The A_(405nm)signal is measured on a Labsystem Multiskan MS microplate reader. Underthe experimental conditions used, the region of linearity is obtainedfor scFv-His6 concentrations of less than 0.8 μg/ml.

[0125] 10—Electrophoresis of Proteins and Western-Blotting Techniques

[0126] SDS-PAGE: The stacking gel is a 4% acrylamide:bisacrylamide(1:29) gel in 125 mM Tris-HCl, pH 6.8; 0.1% SDS, and the separating gelis a 15% acrylamide gel in 375 mM Tris-HCl, pH 8.8; 0.1% SDS. Theelectrophoresis buffer contains 190 mM glycine, 2 mM Tris-base and 0.1%SDS. The gels (8×7 cm) are poured and the electrophoresis is carried outusing a Hoefer Mighty Small II apparatus. 2×loading buffer (125 mMTris-HCl, pH 8.0; 2.5% SDS; 20% glycerol; 2 mg/ml bromophenol blue, and10% 2-mercaptoethanol in the case of reducing SDS gels) is added to thesamples, which are heated for 5 min at 90° C. before being loaded. Theelectrophoresis is performed for 1 h 30 at 30 mA. The markers have thefollowing molecular masses: 21.5; 31; 45; 66.2 and 97.4 kDa (PrestainedMolecular Weight Marker low range, Bio-Rad). The proteins are revealedby staining the gel in a bath of 1 mg/ml Coomassie blue R250, 42%methanol, 16% acetic acid, and then destaining in several baths of 40%methanol, 10% acetic acid.

[0127] Western-blotting technique: the proteins are separated bySDS-PAGE and then transferred onto a Hybond-C nitrocellulose membrane(Amersham) overnight, under 10V, in a chamber consisting of a TE22Series Transphor Electrophoresis Unit HSI (Hoefer), containing 25 mMTris-base, 190 mM glycine, 20% methanol. Next, the membrane is saturatedwith 3% BSA (weight:volume) in TBS buffer (100 mM Tris-HCl, pH 7.5; 150mM NaCl), and then incubated for 1 h at ambient temperature with theprimary and secondary antibodies, diluted in TBS containing 3% BSA, atthe concentrations described for the ELISA (example 1-9). After eachincubation with an antibody, the membrane is washed in two baths of TTTbuffer (200 mM Tris-HCl, pH 7.5; 500 mM NaCl; 0.05% Tween 20; 0.2%Triton X-100) followed by a bath of TBS buffer. The proteins arerevealed by incubating the membrane in 100 mM Tris-HCl, pH 9.5; 100 mMNaCl; 50 mM MgCl₂; 0.4 mg/ml Nitro Blue Tetrazolium (Sigma) and 0.2mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma). The revelation isstopped by immersing the membrane in water.

[0128] 11—Analysis of the Interaction Between mAbD1.3 scFv and henLysozyme Using BIAcore

[0129] The measurements are made on a CM5 chip (BIACORE AB) withcarboxymethyldextran surface, as described in England et al., (1999).Briefly, the lysozyme is immobilized in a channel by amide bonding up toa level of 500 RU (1 RU=1 ng/mm²). The rate constants for association(k_(on)) and for dissociation (k_(off)) at the interface, and also theirK′_(D) (dissociation constant at the interface) ratio, are measured at20° C. with a flow of 10 μl/min of scFv-His6 in PBS buffer supplementedwith 0.005% Tween 20. The buffer also contains 1 μM of lysozyme duringthe dissociation phase in order to avoid re-binding of the scFv-His6s tothe immobilized lysozyme. A channel with no immobilized lysozyme givesthe nonspecific binding signal.

[0130] 12—Exclusion Chromatography

[0131] The scFv-His6 is dialyzed against buffer Q and then concentratedby ultrafiltration through a Centricon^(R) 10 (Amicon). Thechromatographies are performed using a Superdex^(R) 75 HR10/30 column(Pharmacia), in buffer Q, at a flow rate of 0.5 ml/min. The column ispreequilibrated under the same conditions. The proteins are detected inthe effluent of the column using A_(280 nm). The samples, prepared inbuffer Q, are injected using a 200 μl loop. They have the followingcompositions: scFv-His6, 200 μl at 50 μg/ml; BSA, 40 μg;chymotrypsinogen, 14 μg; acetone, 0.1%. The ratio of the dimerconcentration to the monomer concentration is determined from thesurface area of the peaks obtained.

[0132] 13—Coupling of Fluorophores to the Free Cysteine of a MutantscFv-His6

[0133] The mutant scFv-His6s freshly purified and rigorously conservedat 4° C. are dialyzed overnight against buffer C. Alternatively, thepurified mutant scFv-His6s are adjusted to a concentration of at least100 μg/ml by ultrafiltration through a Centricon^(R) 10 filter (Amicon)and then reduced by addition of 2-mercaptoethanol at a finalconcentration of 0.1 mM, 1 mM, 10 mM or 50 mM. The mixture is incubatedfor 1 hour at 22° C. The reduced protein is separated from the excessreducing agent by desalting, using a HiTrap Desalting column (Pharmacia)preequilibrated in highly degassed buffer C.

[0134] The coupling is carried out by addition of NBD chloride, of esterof IANBD, of 5-IAF or of acrylodan (BioProbe) in a molar ratio(protein:fluorophore) of at least 1:20. The mixture is incubated for 24hours at 22° C. and then centrifuged for at least 15 minutes at 10,000g, at 4° C. The labeled scFv-His6 is then repurified. For this, thereaction mixture is diluted 7-fold in 5 mM imidazole in buffer P andsuccessively loaded 3 times onto a column containing 500 μl of Ni-NTAresin. The excess fluorophore is removed by washing 6 times with 5 ml of5 mM imidazole in buffer P. The labeled scFv-His6 is eluted with 1 ml of40 mM imidazole in buffer P and then either with 4 times 500 μl of 100mM imidazole in buffer P or with 3 times 600 μl of 160 mM imidazole inbuffer P. A reference sample is obtained by treating buffer C alone inan identical manner. It is used as a control for the protein assays andthe fluorescence measurements. The coupling yield may be estimated bythe ratio of the fluorophore absorbance to the protein absorbance:

[0135] ε280 nm (scFv-His6)=51.13 mM⁻¹.cm⁻¹, ε435 nm (CNBD)=9.6mM⁻¹.cm⁻¹, ε500 nm (IANBD)=31.1 mM⁻¹.cm⁻¹, ε492 nm (5-IAF)=75 mM⁻¹.cm⁻¹,ε391 nm (acrylodan)=20 mM⁻¹.cm⁻¹, as described in Haugland et al.,(1996).

EXAMPLE 2 Example of Implementation of the Method According to theInvention

[0136] 1—Search for Residues of the Free Fv Fragment of mAbD1.3 Whichcan be Used for Coupling the Fluorophore (FIG. 1)

[0137] The structure of the free Fv fragment of mAbD1.3 and that of thecomplex thereof with lysozyme are known (Bhat et al., 1994). However, inorder to simplify the search for the residues of the Fv which arelocated in the proximity of the active site for binding to the lysozymeand which can be used for coupling the fluorophore, and so as to makethis research applicable to other pairs of macromolecules for which onlythe structure of the complex exists, experimental data regarding thefree Fv of mAbD1.3 were not used. A structural model of the free Fv wasconstructed by deleting the crystallographic coordinates for lysozymefrom the structure of the complex. The structures were analyzed with theWHAT IF series of programs (Vriends, 1990;http://www.sander.embl-heidelberg.de/whatif/). The following principleswere used:

[0138] 1—Three types of residue were sought in mAbD1.3: those which arein direct contact with the lysozyme, those which are in contact via awater molecule, and those for which the solvent accessible surface area(ASA) is modified by the binding of the lysozyme when spheres of radius1.4, 1.7, 2.0, 2.3, 2.6 or 2.9 Å are used for the molecule of solvent.The use of radii greater than that of a water molecule (1.4 Å) makes itpossible to define an enlarged proximity for the antigen at the surfaceof the antibody and to take into account the considerable volume of afluorophore compared to that of a water molecule. The residues ofmAbD1.3 which satisfy one of these criteria, and the nature of thecriterion used, are indicated in columns 1 and 2 of FIG. 1. Columns 8and 9 of FIG. 1 give the nature of the known mutations in mAbD1.3 andtheir effects, in the form of a variation ΔΔG of energy of interactionwith the lysozyme;

[0139] 2—The residues of the receptor which are selected in (1) must bechanged to Cys by mutagenesis, and then coupled to the fluorophore. TheSγ atom of the mutant Cys residue must therefore be exposed to thesolvent in the free Fv, so that it can be attacked by the fluorophoremolecule. It may be supposed, in a first approximation, that the Sγ ofthe mutant Cys residue superposes with the atom in the γ position of thewild-type residue. This atom in the γ position must therefore be exposedto the solvent in the structure of the wild-type free Fv. When an atomis present in the δ position in the wild-type residue, it may mask theatom in the γ position, with respect to the solvent, whereas thismasking will no longer exist after it has been changed to Cys. It hastherefore been considered that the solvent accessibility of an atom inthe δ position may be informative with respect to that of the Sγ of themutant Cys residue. The ASA values for the atoms in the γ and,optionally, δ position, for each residue selected in point 1), are givenin columns 3 and 4 of FIG. 1 (using R=1.4 Å);

[0140] 3—The fluorophore may advantageously be coupled to the Sγ of theCys residue via an aliphatic chain, the length of which varies from 1 to6 carbon atoms for the most common fluorophores, but may be longer.After coupling, the polar and aromatic groups of the fluorophore shouldpreferentially be at the periphery of the interface of contact betweenthe antibody and the antigen, and not within this interface, where theywould be in conflict with the antigen. For this, the aliphatic arm ofthe fluorophore must have access to the solvent in the structure of thecomplex between the labeled antibody and the antigen. In a firstapproximation, this condition is satisfied if one of the groups whichare located in the γ, δ and ε positions in the initial side chain isaccessible to the solvent in the structure of the native complex. TheASA for these groups of side chains is given in the same format as inpoint 2), in columns 5, 6 and 7 of FIG. 1;

[0141] 4—The mutations of the residues of the receptor should not be toodeleterious for the interaction with the antigen if the intention is forthe labeled antibody to maintain a sufficient affinity. Columns 8 and 9of FIG. 1 give the nature of the known mutations in mAbD1.3 and theireffects, in the form of a variation ΔΔG of energy of interaction withthe lysozyme (England et al., 1997 and 1999; Hawkins et al., 1993;Dall'Acqua et al., 1996; Dall'Acqua and Carter, 1998).

[0142] Column 10 of FIG. 1 summarizes the criteria used. The residuesselected are divided into three classes of different priority. The firstclass contains the residues in which the atom in the γ position isaccessible to solvent in the structure of the free Fv, and which are incontact with the lysozyme either directly or via a water molecule:VL-Thr53, VL-Ser93, VL-Thr94 and VH-Thr30. The second class contains theresidues in which the atom in the γ position is accessible and which arenot in direct contact with the lysozyme: VL-Asn31, VL-Thr52, VL-Asp56,VH-Ser28, and VH-Asn56. The third class contains the residues in whichthe atom in the δ position is accessible, but not the atom in the γposition: VL-Asn28, VL-His30, VH-Asp58 and VH-Arg99.

[0143] 2—Construction of scFv-His6 Expression Vectors and Optimizationof the Production of scFv-His6 (FIG. 2)

[0144] a) Construction of scFv-His6 Expression Vectors

[0145] The effect of coupling fluorophores to residues of the scFv ofmAbD1.3 as defined in 1, on the binding to the lysozyme, was studiedusing recombinant mAbD1.3 scFvs having a C-terminal extension of 6histidine residues (scFv-His6), expressed using prokaryotic expressionvectors. The wild-type form and also the mutated forms of scFv wereexpressed and purified on a nickel column.

[0146] Two vectors which encode scFv-His6, pMR1 and pMR5, wereconstructed according to the protocol described in example 1-5. Theycarry the kanamycin resistance gene or ampicillin resistance gene, theM13 phage origin of replication (f1IG), and periplasmic addressingsequences, derived from the pelB or ompA genes, upstream of the scFvgene. The vector pMR5 carries the scFv-His6 gene under the control of aT7 phage promoter and of the lactose operator, in the lactose repressorgene. Expression is carried out in the BL21(DE3) strain which carriesthe T7 RNA polymerase gene under the control of the lacOlacp(UV5)promoter/operator on the λDE3 prophage. The addition of IPTG to theculture medium induces the expression of the RNA polymerase and scFvgenes. The vector pMR1 carries the scFv-His6 gene under the control ofthe tetracycline promoter/operator (tet^(p/o)), and the correspondingrepressor gene. The induction is carried out by adding a nontoxic analogof tetracycline, anhydrotetracycline, which is effective at a very lowconcentration.

[0147] b) Optimization of the Production of scFv-His6

[0148] The production of scFv-His6, estimated by the amount of scFv-His6present in a crude periplasmic extract, is measured by indirect ELISAusing an anti-His5 antibody, according to the techniques described inexample 1-9. This method makes it possible to rapidly test manycombinations of production parameters, such as the culture temperature,the induction time and the inducer concentration (FIG. 2).

[0149] The production of small amounts of antibody fragments from pMR1or from pMR5, under the conditions described in example 1-6, showed thatthe vector pMR1 gave the best yield at 22° C., inducing for 2 hours with0.2 μg/ml anhydrotetracycline. These same conditions were used toproduce mutant scFv-His6s of mAbD1.3 (FIG. 2).

[0150] The production of large amounts of antibody fragments from pMR1or from pMR5, under the conditions described in example 1-6, showed thatthe production yield was better when the induction took place during theexponential growth phase. Now, at 22° C., 2xYT medium allows only ashort exponential growth phase for the HB2151 strain, betweenA_(600 nm)=0.3 and A_(600 nm)=1.1 approximately. In addition, it wasobserved that the bacteria became lysed in this medium. The latter wastherefore supplemented with a weak base (K₂HPO₄) in order to avoidacidification thereof during culturing, and with compounds whichstabilize the outer bacterial membrane (MgSO₄, glucose, NaCl). It wasobserved that, with the resulting medium (SB medium), the exponentialgrowth phase was greatly extended at 22° C., up to A_(600 nm)=3.8. Thismedium made it possible to obtain 4 times more scFv-His6 than in 2xYTmedium, using a given culture volume.

[0151] Two types of method exist for extracting the periplasmic fluidfrom a bacterium: 1) the bacteria are equilibrated in a medium with ahigh osmotic pressure, and then transferred into a hypoosmotic buffer.The osmotic shock causes the outer membrane to rupture and the contentof the periplasm is recovered in the second buffer; 2) the bacteria areresuspended in an outer membrane-lysing buffer (EDTA, amphiphilicpeptides such as polymyxin B). The content of the periplasm is thenrecovered in a single step. Four methods for preparing the periplasmicextract from HB2151(pMR1), described in example 1-7, were tested, andthe amount of scFv-His6 present in each extract was estimated by ELISA.The product of the measurements of A_(405nm) by the volume of theextract gave the following relative values: 100±6 for the osmotic shockmethod (mean±standard error over 3 measurements), 58±9 for the Tris-EDTAmethod, 61±8 for the Tris-EDTA-NaCl method and 106±9 for the polymyxin Bmethod. The latter method gave the best results and was therefore usedfor the productions of wild-type and mutant scFv-His6s in large volume.

[0152] 3—Purification of scFv-His6

[0153] The polyhistidine tail of the scFv-His6s allows them to bepurified on a Ni-NTA immobilized nickel column, according to theprotocol described in example 1-8. After the column has been loaded withthe periplasmic extract, washing and elution are carried out withvarious concentrations of imidazole, which competes with the histidinesfor the nickel coordination. The imidazole concentration for thewashing, which makes it possible to detach the impurities withoutdisplacing the scFv-His6 itself, and the concentration for the elution,which makes it possible to detach the scFv-His6 in a minimal amount offractions without denaturing it, are specified in example 1-8. Thepurity of the elution fractions was analyzed by SDS-polyacrylamide gelelectrophoresis and Coomassie blue staining, according to the protocoldescribed in example 1-10. These fractions contain a protein which hasan apparent molecular mass equal to the expected mass for thescFv-His6s, and are 95% pure. The losses of material due to thenonbinding to the nickel or to the detachment during the washes wereestimated. The product of the measurements of A_(405nm) by the volume ofeach fraction gave the following relative values: of the 100% containedin the crude extract, 6.7% of the scFv-His6s do not bind to the column,7.6% are detached during the washes and 85.6% are in the elutionfractions. Under the optimal conditions for culturing, extraction andpurification, 650 μg of purified wild-type scFv-His6 were obtained perliter of culture.

[0154] 4—Properties of Oligomerization and of Binding to Lysozyme of thewild-Type scFv-His6 (FIG. 4)

[0155] The BIAcore apparatus makes it possible to rapidly obtain therate constants for association k_(on) and dissociation k_(off) betweenantibody and antigen, and also the equilibrium constantK′_(D)=k_(off)/k_(on), at the interface between the buffer and thesurface on which the antigen has been immobilized. Since themeasurements are taken under continuous flow over the surface, they maybe distorted by the recapturing of recently dissociated svFvs on anotherimmobilized antigen site. This recapturing has little influence with amonovalent antibody fragment, and it is limited by adding antigen insolution in the dissociation buffer. However, the scFvs are capable offorming dimers by association of the VH domain of one molecule with theVL domain of another (Arndt et al., 1998). These dimers have twoantigen-binding sites. They lead to an increased probability ofrecapture.

[0156] Before carrying out the measurements on BIAcore, it is thereforenecessary to verify the dimerization state of the scFv-His6 of mAbD1.3.The proportions of monomer and of dimer at the pH used for the BIAcore(pH 7.5) and at a concentration of 50 μg/ml were estimated by the heightof the corresponding peaks in exclusion chromatography, according to theprotocol of example 1-12, and the results are given in FIG. 4. The ratioof the dimer peak surface area to the monomer peak surface area gives adegree of dimerization of 9.8%. This low degree makes it possible todisregard the effect of recapturing on the surface during themeasurements on BIAcore. In addition, these measurements were made atconcentrations of 0.1 to 20 μg/ml, less than the 50 μg/ml used for theexclusion chromatography. The kinetic parameters for the interactionbetween the scFv-His6 and the lysozyme, determined on BIAcore, accordingto the protocol described in example 1-11, are as follows:

k_(on)=1.15(±0.15)0.10⁵M⁻¹.S⁻¹

k_(off)=1.20(±0.22)0.10⁻³S⁻¹

K′_(D)=10.5(±3.3)nM

[0157] 5—Construction, Production and Purification of Mutant scFv-His6s(FIG. 3)

[0158] The mutations to cysteine of the residues of the antibodymAbD1.3, placed in the first class (VH-T30C, VL-T53C, VL-S93C andVL-T94C, see also paragraph 1 of this example and column 10 of FIG. 1)were introduced into the expression vector pMR1 using the Kunkel methoddescribed in example 1-4. The oligonucleotides used, described inexample 1-3, were designed to introduce or delete restriction sites. Themutation can then be detected by analysis of the fragments obtainedafter cleavage. The mutant clones were also sequenced in the region ofthe mutation, using the oligonucleotides described in example 1-3,according to the method described in example 1-4, in order to verify theintegrity of the sequence. The plasmid derived from pMR1 carrying themutation VL-S93C, named pMR1 (VL-S93C), corresponds to SEQ ID NO: 11.The ability of several mutant derivatives of pMR1 to express thescFv-His6 was tested under the conditions described for the productionof the wild-type scFv-His6 from pMR1 (example 2-2). The results showthat these mutants are produced efficiently in comparison with thewild-type: 100±12 for the wild-type, 100±29 for VL-S93C, 97±5 forVL-T94C. The VL-S93C mutant was purified with yields of 575 μg per literof culture, comparable to those obtained with the wild-type scFv-His6.

[0159] The purity of the elution fractions was analyzed bySDS-polyacrylamide gel electrophoresis and Coomassie blue staining,according to the protocol described in example 1-10. The resultsobtained are given in FIG. 3. These fractions contain a protein whichhas an apparent molecular mass equal to the mass expected for thescFv-His6s, and are 95% pure.

[0160] 6—Absence of Covalent Dimerization of the VL-S93C Mutant (FIG. 5)

[0161] The presence of a free cysteine on the mutant scFv-His6s may leadto the formation of interference intermolecular disulfide bridges duringproduction or purification or during coupling of the fluorophore. ThescFv-His6(VL-S93C), dialyzed against the coupling buffer, was analyzedby exclusion chromatography under the conditions used for the wild-typescFv-His6 (example 1-12 and example 2-4). The chromatogram shows onlyone peak, suggesting that the scFv-His6(VL-S93C) exists in only a singlestate of oligomerization in the buffer used (FIG. 5). The position ofthis peak corresponds virtually to that of the monomeric wild-typescFv-His6. The small difference between the positions of these two peaksmay be due to the fact that the two chromatograms were developed inbuffers which were different in nature and had slightly different pH s.

[0162] 7—Coupling of Fluorophores to the VL-S93C Mutant (FIGS. 6 to 10)

[0163] The Coupling of IANBD (FIG. 6) to scFv-His6(VL-S93C) is carriedout after treating the antibody fragment with various concentrations of2-mercaptoethanol and desalting, according to the protocol described inexample 1-13. The reaction is carried out at pH=7.0 in order to avoidinterference coupling on lysines deprotonated at basic pH (Houk et al.,1983; Del Boccio et al., 1991). After coupling, the excess fluorophoreand the scFv-His6(VL-S93C) derivative are separated by furtherpurification on a nickel column. Several arguments converge to confirmthe specificity of the coupling on the cysteine VL-Cys93 of the mutantscFv. During the repurification, the fluorophore is effectively washedand the residual fluorescence is eluted from the column at the same timeas the scFv. In the elution fractions, the fluorescence intensity isproportional to the amount of protein, which suggests stoichiometriccoupling (FIG. 7).

[0164] In the absence of reducing treatment, the wild-type scFv-His6shows virtually no reaction with IANBD, whereas the scFv-His6(VL-S93C)shows a degree of labeling with the fluorophore of 8% (FIG. 8). Thelatter yield is greatly improved when the scFv-His6(VL-S93C) is reducedwith 2-mercaptoethanol, rapidly desalted and placed in the presence ofIANBD. On the other hand, this treatment causes a loss of material whichincreases the more thorough the reduction. It is probable that thescFv-His6 is made partly insoluble by the reduction of its disulfidebridges. The loss of material during the desalting (−30%) andrepurification (−15%) steps also intervenes in the overall couplingyield. A concentration of 10 mM of 2-mercaptoethanol made it possible toobtain satisfactory coupling yields with two different mutants (FIGS. 9and 10), while at the same time preserving sufficient material for thefluorescence measurements.

[0165] 8—Variations in the Fluorescence of the VL-S93C Mutant Coupled toa Fluorophore, as a Function of the Antigen Concentration (FIG. 11 andFIG. 12)

[0166] A spectrum of emission from the scFv-His6(VL-S93ANBD) conjugate,taken immediately after the addition of antigen (lysozyme), shows anenhancement of the fluorescence which depends on the final concentrationof antigen. The fluorescence intensity saturates at 1.26±0.05 times thatof the free biosensor when the antigen concentration is sufficient, andreproducibly over several preparations of biosensors. No significantshift in the wavelength of the emission maximum is observed (FIG. 11).

[0167] The fluorescence intensities were recorded in the absence or inthe presence of varying concentrations of lysozyme (10 nM to 2 μM) in a50 mM Tris-HCl, 150 mM NaCl buffer, pH 7.5.

[0168] The results given in FIG. 12 show that, in this buffer, thefluorescence intensity of the scFv-His6 (VL-S93ANBD) conjugate isproportional to the concentration of lysozyme up to 400 nM and increasedby 91% at saturation. The curve obtained makes it possible to directlytitrate the lysozyme between 10 and 400 nM.

[0169] Consequently, the results obtained in FIG. 12 illustrate that thebiosensor according to the invention advantageously makes it possible todirectly titrate an antigen or a hapten, for example in a body fluid.

[0170] 9—Coupling Yield and Variation in Fluorescence, as a Function ofthe Antigen Concentration, of a Series of scFv-His6 Mutants (FIG. 13)

[0171] ScFv-His6 mutants, selected according to the method described inexample 2-1, were produced in a similar way to the VL-S93C mutant(example 2-5), according to the methods described in example 1 (1-1 to1-12). Thus, plasmids derived from pMR1 (SEQ ID NO: 10) carrying themutations described in FIG. 12 were constructed by site-directedmutagenesis using the oligonucleotides given in table II. The scFv-His6mutants produced from these plasmids were coupled to IANBD, in a similarway to the VL-S93C mutant (example 2-7), using a mercaptoethanolconcentration of 10 mM, according to the protocol described in example1-13. The results given in FIG. 13 show that satisfactory couplingyields are obtained for virtually all the coupling positions selected,and that considerable enhancements of fluorescence are observed for morethan half these coupling positions.

[0172] References

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[0213] As emerges from the above, the invention is in no way limited toits methods of implementation, preparation and application which havejust been described more explicitly; on the contrary, it encompasses allthe variants thereof which may occur to a person skilled in the art,without departing from the context or the scope of the presentinvention.

1 25 1 27 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 1 catctgctaa gcatgttgtataataga 27 2 29 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 2 cgaggagtacaccaaaaatg ttgacagta 29 3 26 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 3cgtccgagga caactccaaa aatgtt 26 4 18 DNA ARTIFICIAL SEQUENCE SYNTHETICDNA 4 agaatattgt gttcctga 18 5 18 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA5 tgctgatgct cagtctgg 18 6 18 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 6ggtgatggaa acacagac 18 7 15 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 7cgccgcgctt aatgc 15 8 43 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 8tcgagatcaa gcggccgctg gaacaccatc accatcacca tta 43 9 43 DNA ARTIFICIALSEQUENCE SYNTHETIC DNA 9 agcttaatgg tgatggtgat ggtggtccag cggccgcttg atc43 10 3922 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 10 acccgacaccatcgaatggc cagatgatta attcctaatt tttgttgaca ctctatcatt 60 gatagagttattttaccact ccctatcagt gatagagaaa agtgaaatga atagttcgac 120 aaaaatctagataacgaggg caaaaaatga aaaagacagc tatcgcgatt gcagtggcac 180 tggctggtttcgctaccgta gcgcaggccg aagttaaact gcaggagtca ggacctggcc 240 tggtggcgccctcacagagc ctgtccatca catgcaccgt ctcagggttc tcattaaccg 300 gctatggtgtaaactgggtt cgccagcctc caggaaaggg tctggagtgg ctgggaatga 360 tttggggtgatggaaacaca gactataatt cagctctcaa atccagactg agcatcagca 420 aggacaactccaagagccaa gttttcttaa aaatgaacag tctgcacact gatgacacag 480 ccaggtactactgtgccaga gagagagatt ataggcttga ctactggggc caagggacca 540 cggtcaccgtctcctcaggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 600 cggacatcgagctcacccag tctccagcct ccctttctgc gtctgtggga gaaactgtca 660 ccatcacatgtcgagcaagt gggaatattc acaattattt agcatggtat cagcagaaac 720 agggaaaatctcctcagctc ctggtctatt atacaacaac cttagcagat ggtgtgccat 780 caaggttcagtggcagtgga tcaggaacac aatattctct caagatcaac agcctgcaac 840 ctgaagattttgggagttat tactgtcaac atttttggag tactcctcgg acgttcggtg 900 gagggaccaagctcgagatc aagcggccgc tggaacacca tcaccatcac cattaagctt 960 gacctgtgaagtgaaaaatg gcgcacattg tgcgacattt tttttgtctg ccgtttaccg 1020 ctactgcgtcacggatctcc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 1080 ggttacgcgcagcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 1140 cttcccttcctttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 1200 ccctttagggttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 1260 tgatggttcacgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 1320 gtccacgttctttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 1380 ggtctattcttttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 1440 gctgatttaacaaaaattta acgcgaattt taacaaaata ttaacgctta caatttcagg 1500 tggcacttttcggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 1560 aaatatgtatccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 1620 gaagagtatgagtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 1680 ccttcctgtttttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 1740 gggtgcacgagtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 1800 tcgccccgaagaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 1860 attatcccgtattgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 1920 tgacttggttgagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 1980 agaattatgcagtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 2040 aacgatcggaggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 2100 tcgccttgatcgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 2160 cacgatgcctgtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 2220 tctagcttcccggcaacaat tgatagactg gatggaggcg gataaagttg caggaccact 2280 tctgcgctcggcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 2340 tggctctcgcggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 2400 tatctacacgacggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 2460 aggtgcctcactgattaagc attggtagga attaatgatg tctcgtttag ataaaagtaa 2520 agtgattaacagcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg 2580 taaactcgcccagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa 2640 gcgggctttgctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg 2700 ccctttagaaggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg 2760 tgctttactaagtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa 2820 acagtatgaaactctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga 2880 gaatgcattatatgcactca gcgcagtggg gcattttact ttaggttgcg tattggaaga 2940 tcaagagcatcaagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc 3000 attattacgacaagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt 3060 cggccttgaattgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtctta 3120 aaagcagcataacctttttc cgtgatggta acttcactag tttaaaagga tctaggtgaa 3180 gatcctttttgataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 3240 gtcagaccccgtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 3300 ctgctgcttgcaaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 3360 gctaccaactctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 3420 ccttctagtgtagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 3480 cctcgctctgctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 3540 cgggttggactcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 3600 ttcgtgcacacagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 3660 tgagctatgagaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 3720 cggcagggtcggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 3780 ttatagtcctgtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 3840 aggggggcggagcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 3900 ttgctggccttttgctcaca tg 3922 11 3922 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 11acccgacacc atcgaatggc cagatgatta attcctaatt tttgttgaca ctctatcatt 60gatagagtta ttttaccact ccctatcagt gatagagaaa agtgaaatga atagttcgac 120aaaaatctag ataacgaggg caaaaaatga aaaagacagc tatcgcgatt gcagtggcac 180tggctggttt cgctaccgta gcgcaggccg aagttaaact gcaggagtca ggacctggcc 240tggtggcgcc ctcacagagc ctgtccatca catgcaccgt ctcagggttc tcattaaccg 300gctatggtgt aaactgggtt cgccagcctc caggaaaggg tctggagtgg ctgggaatga 360tttggggtga tggaaacaca gactataatt cagctctcaa atccagactg agcatcagca 420aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcacact gatgacacag 480ccaggtacta ctgtgccaga gagagagatt ataggcttga ctactggggc caagggacca 540cggtcaccgt ctcctcaggt ggaggcggtt caggcggagg tggctctggc ggtggcggat 600cggacatcga gctcacccag tctccagcct ccctttctgc gtctgtggga gaaactgtca 660ccatcacatg tcgagcaagt gggaatattc acaattattt agcatggtat cagcagaaac 720agggaaaatc tcctcagctc ctggtctatt atacaacaac cttagcagat ggtgtgccat 780caaggttcag tggcagtgga tcaggaacac aatattctct caagatcaac agcctgcaac 840ctgaagattt tgggagttat tactgtcaac atttttggtg tactcctcgg acgttcggtg 900gagggaccaa gctcgagatc aagcggccgc tggaacacca tcaccatcac cattaagctt 960gacctgtgaa gtgaaaaatg gcgcacattg tgcgacattt tttttgtctg ccgtttaccg 1020ctactgcgtc acggatctcc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 1080ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 1140cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 1200ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 1260tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 1320gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 1380ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 1440gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttcagg 1500tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 1560aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 1620gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 1680ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 1740gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 1800tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 1860attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 1920tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 1980agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 2040aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 2100tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 2160cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 2220tctagcttcc cggcaacaat tgatagactg gatggaggcg gataaagttg caggaccact 2280tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 2340tggctctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 2400tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 2460aggtgcctca ctgattaagc attggtagga attaatgatg tctcgtttag ataaaagtaa 2520agtgattaac agcgcattag agctgcttaa tgaggtcgga atcgaaggtt taacaacccg 2580taaactcgcc cagaagctag gtgtagagca gcctacattg tattggcatg taaaaaataa 2640gcgggctttg ctcgacgcct tagccattga gatgttagat aggcaccata ctcacttttg 2700ccctttagaa ggggaaagct ggcaagattt tttacgtaat aacgctaaaa gttttagatg 2760tgctttacta agtcatcgcg atggagcaaa agtacattta ggtacacggc ctacagaaaa 2820acagtatgaa actctcgaaa atcaattagc ctttttatgc caacaaggtt tttcactaga 2880gaatgcatta tatgcactca gcgcagtggg gcattttact ttaggttgcg tattggaaga 2940tcaagagcat caagtcgcta aagaagaaag ggaaacacct actactgata gtatgccgcc 3000attattacga caagctatcg aattatttga tcaccaaggt gcagagccag ccttcttatt 3060cggccttgaa ttgatcatat gcggattaga aaaacaactt aaatgtgaaa gtgggtctta 3120aaagcagcat aacctttttc cgtgatggta acttcactag tttaaaagga tctaggtgaa 3180gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 3240gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 3300ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 3360gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 3420ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 3480cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 3540cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 3600ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 3660tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 3720cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 3780ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 3840aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 3900ttgctggcct tttgctcaca tg 3922 12 28 DNA ARTIFICIAL SEQUENCE SYNTHETICDNA 12 gaggagtacg caaaaatgtt gacagtaa 28 13 28 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 13 catctgctaa ggtacatgta taatagac 28 14 28 DNA ARTIFICIALSEQUENCE SYNTHETIC DNA 14 cagtttacac cacacccggt taatgaga 28 15 28 DNAARTIFICIAL SEQUENCE SYNTHETIC DNA 15 acaccatagc cgcataatga gaaccctg 2816 31 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 16 cttgatggca caccgcatgctaaggttgtt g 31 17 31 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 17catgctaaat aacagtggat attcccactt g 31 18 33 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 18 acaccatagc cagttaagca gaaccctgag acg 33 19 32 DNAARTIFICIAL SEQUENCE SYNTHETIC DNA 19 ttatagtctg tgcacccatc accccaaatc at32 20 31 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 20 atggtacgat ttattaaacattataagggt g 31 21 34 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 21agacgattcc aacaacatat acactggtcc tcga 34 22 35 DNA ARTIFICIAL SEQUENCESYNTHETIC DNA 22 gattccaaca acatatctgg tcctcgactc ctcta 35 23 36 DNAARTIFICIAL SEQUENCE SYNTHETIC DNA 23 tgacacggtc tctcacacta atatccgaactgatga 36 24 28 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 24 caagagtaattggacaatac cacatttg 28 25 32 DNA ARTIFICIAL SEQUENCE SYNTHETIC DNA 25acccttacta aacacaacta cctttgtgtc tg 32

1. A biosensor, characterized in that it consists of (i) at least onefragment of a receptor which is protein in nature, capable of binding toa suitable ligand via an active site, and in which fragment at least oneof its amino acid residues located in the proximity of said active siteis naturally present in the form of a Cys residue, or is substitutedwith a Cys residue, and (ii) a fluorophore coupled to said Cysresidue(s).
 2. The biocaptor as claimed in claim 1, characterized inthat said receptor has one or more disulfide bridges.
 3. The biosensoras claimed in claim 2, characterized in that said receptor is anantibody or an antibody fragment.
 4. The biosensor as claimed in claim3, characterized in that said receptor is a natural or artificialmonoclonal antibody.
 5. The biosensor as claimed in any one of claims 1to 4, characterized in that the fluorophore is selected from the groupconsisting of: IANBD, CNBD, acrylodan, 5-iodoacetamidofluorescein or afluorophore having an aliphatic chain of 1 to 6 carbon atoms.
 6. Thebiosensor as claimed in any one of claims 1 to 5, characterized in thatsaid biosensor is in soluble form.
 7. The biosensor as claimed in anyone of claims 1 to 5, characterized in that said biosensor isimmobilized on a suitable solid support.
 8. The biosensor as claimed inclaim 7, characterized in that said solid support is selected from thegroup consisting of microplates and optical fibers.
 9. A protein-basedchip, characterized in that it consists of a solid support on which atleast one biosensor as claimed in any one of claims 1 to 8 isimmobilized.
 10. A method for preparing biosensors as claimed in any oneof claims 1 to 8, characterized in that it comprises the followingsteps: (a) selecting residues of the receptor by searching for theresidues which, in the receptor-ligand complex, (i) are in directcontact with the ligand, or (ii) are in contact via a water molecule, or(iii) have a solvent accessible surface area (ASA) which is modified bythe binding of the ligand, when use is made of spheres of increasingradius of 1.4 to 30 Å, preferably of 1.4 to 2.9 Å, for the molecule ofsaid solvent; (b) calculating the solvent accessible surface area (ASA),for the free receptor, of the atoms in the γ position and, optionally,in the δ position for each residue selected in (a), using a sphere of1.4 Å, and selecting the residues in which the atom in the γ position orthe atom in the δ position is accessible to the solvent; (c) mutating bysite-directed mutagenesis at least one of the residues selected in (b)to a Cys residue when said residue is not naturally a Cys residue, and(d) coupling the Sγ atom of at least one Cys residue obtained in (b) orin (c) to a fluorophore.
 11. The preparation method as claimed in claim10, characterized in that, in step (b), the ASA values for a residueX_(i) in the i position of the sequence of the receptor are expressed inthe form of percentages of the corresponding ASA values in a tripeptideGly-X_(i)-Gly, which would adopt the same configuration as thetripeptide X_(i−1)-X_(i)-X_(i+1) in the structure of the receptor. 12.The preparation method as claimed in claim 10 or claim 11, characterizedin that, prior to step (a), it comprises a step of modeling the receptorand/or the ligand and/or the receptor-ligand complex.
 13. A method forpreparing biosensors as claimed in any one of claims 1 to 8,characterized in that it comprises the following steps: (a₁) identifyingthe active site of the receptor by mutagenesis of the set, or of asubset, of the residues of the receptor, and determining the variationsin the parameters of interaction with the ligand (K_(D), k_(on),k_(off)) which are due to each mutation or to limited groups ofmutations; (b₁) selecting the Cys residues, or the residues to bemutated to cysteine, from the residues of the receptor which are locatedin the proximity of the residues of the active site along the sequence;(c₁) mutating by site-directed mutagenesis at least one of the residuesselected in (b₁) to a Cys residue when said residue is not naturally aCys residue; and (d₁) coupling the Sγ atom of at least one Cys residueobtained in (b₁) or in (c₁) to a fluorophore.
 14. The preparation methodas claimed in any one of claims 10 to 13, characterized in that, priorto step (a) or to step (a₁), the nonessential Cys residues of thereceptor are substituted with Ser or Ala residues by site-directedmutagenesis.
 15. The preparation method as claimed in any one of claims10 to 14, characterized in that, in step (d) or in step (d₁), saidfluorophore is selected from the group consisting of IANBD, CNBD,acrylodan, 5-iodoacetamidofluorescein or a fluorophore having analiphatic chain of 1 to δ carbon atoms.
 16. The preparation method asclaimed in any one of claims 10 to 15, characterized in that, prior tostep (d) or to step (d₁), the mutated receptor obtained in step (c) orin step (c₁) is subjected to a controlled reduction.
 17. The preparationmethod as claimed in any one of claims 10 to 16, characterized in that,after step (d) or step (d₁), it comprises an additional step (e) or (e₁)for purifying the biosensor.
 18. The preparation method as claimed inclaim 17, characterized in that, after step (e) or step (e₁), itcomprises an additional step for measuring the equilibrium constant(K_(D) or K′_(D)) for said purified biosensor, or the dissociation(K_(off)) and association (k_(on)) rate constants for the receptor andligand.
 19. The preparation method as claimed in any one of claims 10 to18, characterized in that, after step (d) or (d₁) or step (e) or (e₁),it comprises an additional step for immobilizing the biosensor on asolid support.
 20. The use, in vitro, of the biosensors as claimed inany one of claims 1 to 9, for applications comprising detecting,assaying and locating ligands.
 21. The use of biosensors as claimed inany one of claims 1 to 9, for screening protein libraries.
 22. The useof biosensors as claimed in any one of claims 1 to 9, for sortingmolecules.
 23. The use of the biosensors as claimed in any one of claims1 to 9, for sorting cells.
 24. The use of the biosensors as claimed inany one of claims 1 to 8, for producing protein-based chips.
 25. The useof the plasmid pMR1 of sequence SEQ ID NO: 10, deposited with theCollection Nationale de Culture de Microorganisms (CNCM), under thenumber I-2386, dated Feb. 29, 2000, or of derivatives thereof, forpreparing a biosensor as claimed in any one of claims 1 to
 8. 26. Theuse of the plasmid pMR1 (VL-S93C) of sequence SEQ ID NO: 11, depositedwith the Collection Nationale de Culture de Microorganisms (CNCM), underthe number I-2387, dated Feb. 29, 2000, derived from the plasmid pMR1 ofsequence SEQ ID NO: 10 as claimed in claim 25, for preparing a biosensoras claimed in any one of claims 1 to
 8. 27. A reagent for detecting,assaying or locating ligands, characterized in that it includes at leastone biosensor as claimed in any one of claims 1 to
 9. 28. A method fordetecting, assaying or locating a ligand in a heterogeneous sample,characterized in that it comprises bringing said heterogeneous sampleinto contact with at least one reagent as claimed in claim
 27. 29. A kitfor detecting, assaying or locating ligands, characterized in that itincludes at least one reagent as claimed in claim
 27. 30. A kit forscreening for inhibitors of the ligand/receptor interaction,characterized in that it includes at least one reagent as claimed inclaim
 27. 31. The plasmid pMR1, deposited with the Collection Nationalede Culture de Microorganismes (CNCM), under the number I-2386, datedFeb. 29,
 2000. 32. The plasmid pMR1 (VL-S93C), deposited with theCollection Nationale de Culture de Microorganismes (CNCM), under thenumber I-2387, dated Feb. 29, 2000.